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The methods for isolation of single cells from the guinea pig longitudinal ileum were investigated with focussing on the papain concentration for the digestion of the ileum. The ileal muscle was minced. The minced muscle was loaded with fluorescent probes of calcein or fura 2, and treated with papain for 30 min at various concentrations. Papain at concentrations more than 1 U/ml reduced both calcein fluorescence and fura-2-signal evoked by carbachol. Carbachol-induced fura-2-signal was more sensitive to papain than calcein fluorescence, suggesting that proteins related to the formation of receptors are more vulnerable than membrane lipids or proteins limiting the membrane permeability upon the exposure to papain. The resultant yield of single cells was highest at 0.56 U/ml of papain without affecting calcein and fura-2 fluorescence responses, thus this concentration appeared to be appropriate for the isolation of single cells from the ileum. Single cells alive contracted dose dependently by the exposure to carbachol (0.1-10 microM) under the microscopic measurement, and were appeared to grow confluent in culture for approximately 15 days. These results suggest that the low concentration, 0.56 U/ml, of papain in the isolation medium is better to obtain functional cells from the guinea pig ileum.

Effects of Papain on Isolation of Single Smooth Muscle Cells from the Guinea Pig Longitudinal Ileum.